Chronic idiopathic urticaria (CU) is defined as recurrent hives occurring for at least 6 weeks. In the majority of cases, there is no identifiable trigger, despite extensive evaluation for an underlying etiology. A subset of these patients is classified as having autoimmune urticaria defined by the presence of a functional IgG antibody to the alpha-subunit of the high-affinity IgE receptor (FcεRIα) or to IgE (Kaplan, 2004). These antibodies trigger mast cell and basophil degranulation by the engagement of this receptor. Functional IgG antibody to the receptor has been identified in approximately 30-40% of patients with CU, and anti-IgE antibody has been identified in another 5-10% of patients. Non-functional antibodies to FcεRI may be found in other autoimmune conditions (Fiebiger, 1998).
Techniques to detect the autoantibody to FcεRIα include Western blot and enzyme linked immunoabsorbant assay (ELISA), which are technically time consuming and fail to identify antibodies with histamine releasing properties. Detection methods for functional antibodies include the autologous serum skin test (ASST) and basophil histamine release (BHR). The ASST involves an intradermal injection of the patient's serum into the skin with observation for a wheal and flare reaction. ASST is approximately 70% sensitive and 80% specific when compared with the basophil histamine release (BHR) assay (Aabroe, 1999).
Recently, flow cytometry has been used to identify activated basophils in both allergic disease and chronic urticaria. Upregulation of three proteins (CD63, CD203c, and CD69) on peripheral blood basophils from patients with CU has been described (Vasgar, 2003), although no correlation with autoimmune CU or histamine release was described for any of these markers.
With regard to CD63, two studies have been published demonstrating that sera of patients with CU and positive ASST induce higher expression of CD63 when compared to skin test negative chronic urticaria sera (Wedi, 2000; Gyimsi, 2004). CD63, a member of the transmembrane-4 superfamily, is a basophil and mast cell activation marker that is expressed as a result of the fusion between intracytoplasmic granules and the plasma membrane. It has been proposed that it rapidly appears on the basophil surface upon the addition of anti-IgE, allergen or IL-3 (Knol, 1991; Ebo, 2004). However, CD63 is not specific to basophils and mast cells, and can be expressed on other cells present in the peripheral blood, i.e., monocytes and platelets (Buhring, 2004).
Specifically, Wedi et al. (Wedi, 2000), who used interleukin-3 (IL-3) to activate cells in whole blood, found increased CD63 expression in 70% of ASST positive CU patients, in 45% of ASST negative CU patients, and in 35% of controls. Wedi et al. was not able to demonstrate specificity of the assay for patients with CU and the functional positive ASST, and could not correlate CD63 expression with histamine releasing activity, nor could Wedi et al. show that CD63 expression was characteristic of patients with ASST+sera or with CU patients in general (noting the high percentage of negative controls with increased CD63 expression). Gyimesi et al (Gyimesi, 2004) attempted to improve upon the results of Wedi et al. by using donor basophils from highly sensitized atopic individuals as the test cells. Gyimesi et al. found elevated CD63 expression induced by 11/12 ASST positive sera (92%) and 3/18 (17%) of ASST negative sera. However, the use of the highly sensitized atopic donor cells appears to be a requirement for the improve sensitivity of this test.
Therefore, there remains a need in the art for a rapid, in vitro assay that is minimally invasive, and is capable of distinguishing autoimmune patients from patients with other forms of urticaria, including other forms of chronic urticaria, such as that caused by allergy (atopic patients).